PROJECT SUMMARY Biofilm bacteria cause two-thirds of infections in modern clinical practice, including wound and device- related infections. Host defenses and most available antibiotics are inactive against biofilms, rendering the infections they cause challenging to treat. Given the failure of antibiotics in management of biofilm-associated infections, novel and innovative approaches are needed. Avoiding antibiotics will also decrease the dysbiosis and selection of genotypic antibiotic resistance linked to their use. We are developing electrical anti-biofilm strategies. In the original funding period, we developed the ?electricidal effect,? which uses fixed direct current (DC). Though fixed DC exhibited activity in vitro and in animal models of orthopedic foreign body infection, concerns about mechanistic non-specificity, and challenges in clinical delivery for orthopedic infections (at least as an initial application), led us to propose a new approach. Here, we propose a mechanistically-precise electrochemical strategy (distinct from fixed DC), that will specifically deliver one or the other (or both) of two anti-biofilm chemicals ? hydrogen peroxide (H2O2) and/or hypochlorous acid (HOCl). We have also moved to a more clinically feasible target ? wound infections. Our new approach employs a completely novel, tunable ? potential-controlled ? electrochemical system, which will cleanly deliver continuous low concentrations of H2O2 and/or HOCl, suitable for biofilm killing, without compromising wound healing. Importantly, both chemicals have applications in wound care, but use has been hindered by rapid decomposition of their raw chemical forms. To bring together the biology, microbiology, electrochemistry, engineering and animal model expertise required for our studies, we are newly collaborating with an electrochemist and biofilm engineer, Haluk Beyenal, PhD. In collaboration, we will develop scalable electrochemical bandages (e-bandages) composed of carbon fibers that will generate sustained, controlled quantities of H2O2 and/or HOCl in the presence of specific applied potential, providing an easy-to-use, antibiotic-free approach for treatment of infected wounds and promotion of wound healing. The technology being applied was not available in the prior funding period. We have built prototype devices that generate continuous, controlled concentrations of H2O2 or HOCl (based on applied potential) in amounts that reduce biofilms. Both H2O2- and HOCl-generating prototypes reduced Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus biofilms in vitro. Further, the HOCl-generating prototype reduced the number of live bacterial cells of all three species on porcine dermal explants, and the H2O2-generating prototype did the same against A. baumannii on explants, without damaging the animal tissue. In Aim 1, we will build e-bandages and confirm, using in vitro studies, their biofilm treatment properties, tuning them to ensure lack of toxicity. In Aim 2, we will demonstrate activity and safety of the e-bandages in a murine wound infection model. Our goal is to have a product ready for human testing at the end of our studies.